Cellular transplantation is a recently developed biomedical technology for the study and treatment of human diseases characterized by cell dysfunction or cell death. For many such diseases current medical therapies or surgical procedures are either inadequate or nonexistent. Cellular therapy can replace or augment existing tissue to provide restorative therapy for these conditions. Exemplary cell types suitable for transplantation include: neural tissue derived cells, hepatocytes. myocytes, retinal cells, endocrine cells, melanocytes, keratinocytes, and chondrocytes. It has been shown in both animal models and in human studies that engraftment of transplanted cells can successfully reestablish tissue function.
In a specific example, cells derived from neural tissue have been used to affect the course of Parkinson""s disease, a disorder characterized by depletion of dopaminergic neurons. In neurotoxin-induced, dopamine deficient monkey and rat animal models, xenogeneic and allogeneic fetal ventral mesencephalon (VM) cell preparations isolated from pigs, rats, or humans have been reported to reverse the movement disorder (see e.g., Kopyov et al. 1992 Transplantation Proceedings 24: 547-548;, Huffaker et al. 1989 Exp. Brain Res. 77:329). Human fetal VM grafts have also been reported to affect the course of Parkinson""s disease in man by reinervating the dopamine (DA) depleted host striatum (see e.g., Lindvall et al. 1990 Science, 242: 574-577; Lindvall et al. 1994 Ann. Neurol. 35: 172-180; Widner et al. 1997 Ann. Neurol. 42:95.; Kordower et al. 1995 New Engl. J. Med. 332:1118).
Nevertheless, current methods of cell transplantation, particularly those which utilize freshly prepared neural tissue, have been hindered by the lack of available cell sources and limited viability of neural cells after preparation. These problems are compounded by the logistical problems involved in ensuring that surgeons, operating rooms, patients, and fresh cells are all available at the same time. Finally, the need to rapidly implant the fresh cells following preparation hinders extensive quality control prior to implantation.
In view of the above, it is desirable to store, and sometimes pool, freshly harvested cells prior to implantation. It would also be desirable to store cells which have been cultured in vitro. Such storage would allow banking, quality control, and other desired procedures and manipulations, either in connection with in vitro analysis or implantation in vivo.
Methods for cell storage prior to transplantation include preserving the tissue by freezing cells (xe2x80x9ccryopreservationxe2x80x9d) (see e.g., Chanaud et al. 1987 Neurosci. Lett. 82 : 127-133; Collier et al. (1987) 436 : 363-366) or by refrigerating the cells at above freezing temperatures (xe2x80x9chibernationxe2x80x9d) (see e.g., Sauer et al. 1991 Neurology and Neuroscience 2 : 123-135; Gage et al. 1985 Neurosci. Lett. 60 : 133-137). However, freezing of fresh neural tissue results in poor viability after thawing and low yield or recovery of cell numbers. Specifically, studies comparing survival of cryopreserved and fresh transplanted tissue have shown dramatic decreases in the survival of cryopreserved grafts as compared to fresh control tissue (see e.g., Jensen et al. 1984 J. Comp. Neurol. 227 : 558-68). For example, tissue culture experiments have indicated that cryopreservation may lead to decreases in neuronal survival to between one-and two-thirds of fresh control values (see e.g., Collier et al. 1988 Progress in Brain Research, 78 : 631-6). In particular, human tissue may be more susceptible to damage induced by freezing than rat tissue, which is reflected in poor graft survival and reduction in cell size of the cryopreserved neurons (Frodl et al. 1994 Brain Research, 647:286-298).
Storage of tissue in preservative media at temperatures above freezing temperatures (hibernation) can result in high rates of graft survival and function as compared to cryopreserved tissue (see e.g., Sauer et al. 1991 supra.: Kawamoto et al. 1986 Brain Res., 384 : 84-93). However, cells cannot be maintained for long periods of time under such conditions. Specifically, cell viability is progressively decreased during hibernation. Within about one week, such losses render the cell population unacceptable for transplantation in vivo.
In addition, prior art methods for freezing and hibernating cells utilize complex media comprising buffers and added protein, sometimes including entirely undefined components, such as serum. However, to minimize toxicity and immunogenicity such additives are not desirable for transplantation into humans and hinder controlled studies of neural cell growth, development and function in vitro.
This invention solves the problems referred to above by providing methods for storing neural cells without significant decreases in cell viability and/or functionality. Such methods greatly enhance the availability of cells for in vitro analysis and/or transplantation in vivo. Such methods are also useful when pooling of cells is desired.
In one aspect. the invention pertains to a method for storing cells in a cryopreserved state in which fresh or cultured neural cells are suspended in a cryopreservation solution, the temperature of the cell suspension is decreased in a controlled manner to about xe2x88x92196xc2x0 C. and the cells are maintained in a frozen state.
In another aspect, the invention pertains to a method for storing cells in hibernation in which fresh, cultured, or cryopreserved neural cells are suspended in a hibernation medium which is preferably free of added protein, free of a buffer, or free of added protein and a buffer, and the cell suspension is maintained at temperatures which are above freezing and sufficiently below normal body temperature such that normal physiological cell processes are decreased or halted.
In another aspect, the invention pertains to cultures of cells which have been stored according to a cryopreservation method and/or hibernation method of the invention. Such cells are useful for in vitro growth, development, and analysis as well as for transplantation in vivo.
In another aspect, the invention pertains to methods of implantation which utilize neural cells which have been stored according to the methods disclosed herein.